1. Technical Field
Agents that inhibit urea transport activity are needed as therapeutic agents for increasing solute clearance in states of fluid overload and for treating diseases and conditions such as hypertension. Agents that inhibit urea transporters and methods for using these agents are described herein.
2. Description of the Related Art
Urea is generated by the liver as the major end product of nitrogen metabolism, released into the blood, and excreted by the kidneys. The processing of urea by the kidney is complex, involving countercurrent multiplication and exchange mechanisms that greatly increase urea concentration in the renal medulla compared to serum. In the maximally concentrating (antidiuretic) kidney, urea concentration in the urine can reach >1000 mM in mammals (see, e.g., Bankir et al., “Urea and the kidney” In: The Kidney. 6th ed., edited by Brenner B M, Philadelphia, (WB Saunders, 2000), 637-679; Sands et al., Semin Nephrol 29:178-95, 2009) much greater than the serum urea concentration of 4-10 mM. The renal countercurrent mechanisms involve intrarenal urea recycling facilitated by urea transporters (UTs) expressed in renal tubule epithelial cells (UT-A, encoded by the SLc14A2 gene) and renal vasa recta microvessels (UT-B, encoded by the SLc14A1 gene) (see, e.g., Bagnasco, Am J Physiol Renal Physiol 284: F3-F10, 2003; Sands, Curr Opin Nephrol Hypertens 13:525-32, 2004; Shayakul et al., Pflugers Arch 447: 603-609, 2004; Stewart, Br J Pharmacol 2011 Mar. 30, doi:10.1111/j.1476-5381.2011.01377.x Epub ahead of print; Tsukaguchi et al., J Clin Invest 99:1506-15, 1997. Phenotype analysis of knockout mice lacking UT-B (see, e.g., Bankir et al., Am J Physiol Renal Physiol 286:F144-F151, 2004; Yang et al., J Biol Chem 277:10633-37, 2002) or various UT-A isoforms (see, e.g., Fenton et al., Proc Natl Acad Sci U.S.A. 101:7469-74, 2004; Fenton et al., J Am Soc Nephrol 16:1583-92, 2005; Uchida et al., Mol Cell Biol 25: 7357-63, 2005) has provided evidence for the involvement of UTs in the urinary concentrating mechanism, subject to the caveat that gene knockout may produce off-target effects such as compensatory changes in the expression of non-UT transport proteins (see, e.g., Fenton, Curr Opin Nephrol Hypertens 17:513-18, 2008; Klein et al., J Am Soc Nephrol 15:1161-67, 2004). Though UT function has been studied mainly in kidney, UTs are also expressed in erythrocytes, testis, brain, heart and urinary bladder (see, e.g., Doran et al., Am J Physiol Regul Integr Comp Physiol 290:R1446-R1459, 2006) where their physiological functions are not clear.
Phenotype analysis of mice separately lacking vasa recta UT-B or inner medullary collecting duct UT-A1/3 implicated UT involvement in the formation of concentrated urine and in renal urea clearance (see, e.g., Yang et al., J. Biol. Chem. 277:10633-37 (2002); Fenton et al., Proc. Natl. Acad. Sci. USA 101:7469-74 (2004); Fenton et al., J. Am. Soc. Nephrol. 16, 1583-92 (2005)). The UT-B knock-out mice that were generated manifested a urea-selective urinary concentrating defect associated with urinary hypoosmolality and increased renal urea clearance (Yang et al., supra). UT-B is also expressed outside of the kidney, most notably and in highest abundance in red blood cell (RBC) membranes. Loss-of-function human UT-B mutations result in greatly reduced urea permeability in RBC and a mild urinary concentrating defect (Sands et al., J. Am. Soc. Nephrol. 2:1689-96 (1992); Lucien et al., J. Biol. Chem. 273:12973-80 (1998)).
Diuretics are administered widely in humans to increase renal salt and water clearance in a variety of conditions that are associated with total body fluid overload, such as congestive heart failure and cirrhosis, as well in normovolemic states such as hypertension and syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Most diuretics are inhibitors of salt absorption by kidney tubules, such as a furosemide block of Na+/K+/2Cl− co-transport in the thick ascending limb of Henle and a thiazide block of Na+/Cl− co-transport in the distal tubule. Recently, a new type of diuretic, called an “aquaretic,” has been developed to increase renal water clearance in hyponatremia associated with fluid overload or SIADH (see, e.g., Goldsmith, Am. J. Cardiol. 95:14B-23B (2005); Miller, J. Am. Geriatr. Soc. 54:345-53 (2006)). Vasopressin-2 receptor (V2R) antagonist aquaretics have been approved for clinical use in some countries, though not yet in the United States, and aquaporin inhibitor aquaretics are under development.
Functional studies in knock-out mice indicate a critical role for urea transporters (UTs) in the urinary concentrating mechanism and in renal urea clearance. However, potent and specific urea transport blockers have not been available. Accordingly, a third type of diuretic is needed: one that targets renal urea clearance mechanisms. Because urea is of at least equal importance to NaCl in the renal countercurrent mechanism for urinary concentration (see, e.g., Bankir et al., supra; Masilamani et al., In The Kidney (6th Edition), Brenner, ed. Philadelphia, Pa.; WB Saunders Company; pages 595-35; (2000)), such diuretics are needed for increasing solute clearance in states of fluid overload, hypertension, and may also be useful in prolonging dialysis-free survival in chronic renal insufficiency.
Urea transporter inhibitors identified to date have included non-selective membrane intercalating agents, urea analogs with insufficient potency, and specific urea transporter inhibitors with lower than desired potency. A need exists in the medical art for compounds that inhibit urea transporter and that exhibit nanomolar potency for increasing solute clearance and free water excretion in states of fluid overload, hypertension, and chronic renal insufficiency.